One of the most frequently mutated sites found in human tumors is the INK4a/ARF locus located on chromosome 9. This locus encodes the two tumor suppressor proteins p16 INK4a and p14 ARF. Although the function of both proteins in regulating the activities of Rb and p53 is well understood, their contribution in preventing cancer in humans is not. The goal of this proposal is to better understand how loss of p14ARFand/or p16 INK4a function contributes to the formation of tumors in humans. To disrupt p14 ARF and p16 INK4a activity, homozygous knockout cell lines will be generated by targeted homologous recombination in non-transformed, normal human diploid fibroblasts. Targeting vectors will be generated that will specifically disrupt the function of p14ARFand p16 INK4a while leaving the other member of the INK4a/ARF locus intact and functional. The homozygous knockout cell lines will be analyzed on the macroscopic level for proliferation defects and on the molecular level in order to understand and interpret the phenotypic changes observed. The experiments proposed will also determine whether disruption of the p53- and/or the Rb-pathway is required for neoplastic transformation of normal human diploid fibroblasts. p21-/-/p16 INK4a-/-double knockout cell lines will be generated to analyze how the Rb- and p53-pathways are connected on a molecular level and how they cooperate in the prevention of neoplastic transformation of human cells. The data gathered will significantly advance our understanding of how mutations in the INK4/ARF locus contribute to formation of tumors in humans.